The overall goal of the following experiment is to develop a new technique for analyzing microcircuitry in the central nervous system. This is achieved by first defining a population of neurons for infection by expression of the TVA receptor. As a second step, the trans synaptic virus is injected into the CNS, which infects the primary neurons of choice.
Next, the virus will only spread trans synaptically to connected neurons that express the TVA receptor. In order to examine if these neurons are connected, results are obtained to show that this technique can be used to analyze microcircuitry based on the validation of labeling known connected microcircuitry. The main advantage of this technique over existing methods like electrophysiology and microscopy, is that it is a high throughput method for analyzing neuronal circuitry.
We first had the idea for this method when we read the first study of mono synaptically restricted viral tracing by wickersham and colleagues. We felt that viruses could be further engineered to study connected neurons in novel ways. Please consult the written protocol for details on recovery of VSV from CD NA using the VACCINIA T seven system to generate cells for passaging.
VSV first aspirate the media from four 80%cofluent 10 centimeter plates of TV eight 800 cells plated the previous day and replaced with five milliliters of unsu. Supplemented DMEM transfect each plate with five micrograms of the plasmid, encoding the glycoprotein of choice according to standard procedures. After about six hours post transfection replace media with DMEM supplemented with 10%FBS incubate the plates overnight in a tissue culture incubator set to 34 degrees Celsius with 5%carbon dioxide to allow glycoprotein expression and surface accumulation.
Following the incubation, aspirate the media and replace with five milliliters of DMEM plus 10%FBS then infect each plate with VSV containing the MA rabies G chimeric glycoprotein at a multiplicity of infection of 0.01 to 0.1 and incubate at 34 degrees Celsius overnight 24 hours later. Check the infection levels of each plate by fluorescence microscopy. If more than 50%of cells were infected, collect the media.
Replace with five milliliters of fresh media and then freeze the collected media at minus 80 degrees Celsius. Repeat the collection procedure 24 hours thereafter for three days for a total of four days of collection. Freezing each aliquot of collected media after the final aliquot has fully frozen at minus 80 degrees Celsius, though all of the collected media aliquots at 37 degrees Celsius at this point, there should be 80 milliliters of collected media past the collected media through 0.45 micron filters on ice.
Then aliquot 36 milliliters of virus into a Beckman centrifuge tube, and concentrate the supernatant by ultracentrifugation at 80, 000 times G for 90 minutes at four degrees Celsius. Make sure that the rotor is properly balanced following the centrifugation to count into a peaker containing 10%bleach and use an aspirator to remove liquid from the side of the tube. Then shake the tubes containing the virus at 120 RPM for one hour at four degrees Celsius and resuspend the viral pellet in the smallest volume possible, which is usually around 30 microliters.
Once the virus is resuspended tighter the virus by infecting a 24 well plate of 2 9 3 T cells with a one to 1 million tenfold dilution series and titering about two days post-infection. According to standard procedures, virally infected cells can be detected by fluorescence. It's important to use the correct moline for these experiments.
The goal is to have the TVA receptor expressed in the cell types into which one wants to map. This can be achieved by expression of the TVA receptor by viral means or through a transgenic allele. This experiment uses six to 10 week old triple transgenic mice.
Mice expressing a conditional TVA allele will crossed a mice expressing a chat, Cree allele. Then these mice were crossed with a line that express a fluorescent TD tomato allele. The final transgenic mouse expresses TVA and a conditional TD tomato allele in all cells expressing Cree, including the starburst omicron cells of interest.
And this expression can be visualized using fluorescence microscopy. Check the stereotaxic coordinates for the region of interest in a mouse brain atlas and prepare the stereotaxic apparatus by turning on the micro injector controller and moving the plunger and electrode holder into place. Then backfill the needle with mineral oil to provide an interface with the virus and place the needle on the plunger ensuring that there are no air bubbles in the needle.
Haw the virus and place it on ice. Set the micro injector to withdraw and move the tube virus underneath the capillary on the micro injector. Lower the tip of the capillary into the virus and withdraw the necessary amount of virus for the experiment.
After a preoperative dose of buprenorphine is given, the animal is appropriately anesthetized according to institutional guidelines. Perform a toe pinch and ensure that the reflex response is absent. Before beginning the procedure.
Place the anesthetized mouse on a heating pad propped on a movable stand for positioning the mouse. Apply eye ointment to prevent drying of the cornea while the animal is anesthetized. Now, place the mouse in the stereotaxic apparatus by gently inserting the ear barss into the air canal.
Ensure that the head is parallel to the base of the stereo attacks and does not move laterally. Next, adjust the bite bar to help stabilize the head. Remove the fur in the area of the incision.
Then wash this area three times with ethanol, followed by three times with iodine. After making a sagittal incision in the skin with a pair of scissors to reveal the skull, push back the skin with homemade surgical hooks. Then learn up the micro capillary with bgma and use the dials to move the micro injector to the coordinates for the lateral gent nucleus.
Once the LGN has been located, drill a small hole in the skull over the site of injection. Position the capillary over the hole and slowly lower it to a position just dorsal of the brain surface. This is the zero position of the dorsal ventral coordinate.
The micro capillary should be sharp enough to puncture the mouse dura. So slowly lower it to a depth of 2.75 millimeters and inject the required amount of virus at a speed of around 100 liters per minute. Allow the needle to remain in the brain for about seven minutes to permit diffusion of the virus from the injection site and prevent the virus from moving up the needle.
Tracted then very slowly over a period of two minutes, raise the needle from the injection site and outta the brain. Finally suture the surgical site and monitor the animals until they have recovered from anesthesia. Administer buprenorphine every 12 hours for two days.
Generally, VSV trans synaptic spread can be observed around 24 hours post-injection. However, longer waiting times will result in more spread. After euthanizing the mouse using carbon dioxide inhalation, dissect out the eye into 4%formaldehyde and incubate for one hour, ensuring that the eye remains submerged.
Then transfer the eye to a Petri dish containing PBS. Use a pair of number 55 forceps to quickly remove the cornea and loosen and dissect the retina away from other tissues. The whole mount analysis.
Cut the retina on a glass slide so that it lays flat. Place silicon spaces around the edges of the glass slide cover with prolonged gold mounting media and then cover slip using a zero thickness glass cover slip for analysis of retina sections. Place the retina in 30%sucrose in PBS until the retina sub merges.
Then place the retina in a mixture consisting of a one-to-one ratio of OCT to 30%sucrose solution for three hours. Finally, flash, freeze the retina and store at minus 80 degrees Celsius until ready to section. This image shows a time course experiment demonstrating infectivity of TVA 800 cells and 2 9 3 T cells by vsv A slash rg.
The upper panels show TVA 800 cells at six hours one day and two days post infection. The lower panels show 2 9 3 T cells at the same time points. The virus spreads between TVA expressing cells in a matter of hours, whereas the spread on 2 9 3 T cells, which do not express the TVA receptor, though it does occur, happens much slower.
The scale bar represents 100 microns. This image shows a representative infection of retinal ganglion cells of non TVA expressing mice. Animals were injected into the LGN with the vsv A slash RG virus.
Pseudo typed with R-A-B-V-G and tissue was harvested two days post-injection. A sparse infection is seen here with the retinal ganglion cells indicated by yellow arrowhead. This image shows a denser infection.
This can be achieved with a higher TI of virus and a good quality injection as seen here. Different types of retinal ganglion cells can be identified based on morphology including small arbor ganglion cells, type one melanopsin ganglion cells on direction selective ganglion cells or DS GCs and on off DS GCs. Here the scale bar represents 50 microns as seen in the following images from A TVA expressing mouse.
VSVA slash RG transmission from on-off DS GCs to Starburst omics follows the expected pattern. The on-off DS GCs indicated with white arrowhead, with green fill transmit virus to multiple starburst omics indicated by yellow arrowhead. As seen here all green cells that are not DS GCs co label with the Cree reporter indicating that they are TVA expressing Starburst omics.
The DS GCs indicated by white arrow heads with green fill and starburst omics indicated by yellow arrow heads stratifying the appropriate chat layers of the retinal inner plexiform layer or IPL, the cells to which the virus transmits include both on and off. Starburst omicron. The numbers in this figure indicate the layers of the IPL Once mastered, the entire experiment can be done in one or two days if it's performed properly.
The speed of VSV is one of its major advantages following this procedure. Other methods like channel red, opsin based circuit mapping can be performed in order to validate the viral circuitry tracing and to analyze the properties of these synaptic connections. After watching this video, you should have a good understanding of most of the different steps involved in viral circuit tracing from growing virus to in vivo injection, to analyzing fluorescently labeled neurons.
